CN112525870A - Large-area fluorescence imaging detection device - Google Patents
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Abstract
The application discloses a large-area fluorescence imaging detection device, which comprises a fluorescence excitation light path, an excitation light reflection light path and a fluorescence projection light path, wherein the fluorescence excitation light path comprises an excitation light source component, a collimating lens group, an excitation light filter assembly and a dichroic mirror which are sequentially arranged; the exciting light reflection light path comprises a light limiting hole plate and an objective table which are arranged in sequence; the fluorescence projection optical path comprises a light limiting hole plate, a dichroic mirror, a fluorescence optical filter assembly, an optical imaging lens and an image sensor which are sequentially arranged; the light rays entering the dichroic mirror form an angle of 45 degrees +/-10 degrees with the mirror surface of the dichroic mirror, and the light rays reflected by the dichroic mirror form an angle of 0 degrees +/-10 degrees with the normal line of the objective table; the exciting light reflection optical path is coaxial with the fluorescence projection optical path. The large-area fluorescence imaging detection device disclosed by the application has the characteristics of small volume, low cost, simplicity in operation and high light source energy utilization rate, and realizes large-area fluorescence imaging detection aiming at biochips, digital PCR single-layer tiled micro-droplet arrays and the like.
Description
Technical Field
The application relates to the technical field of fluorescence imaging detection in molecular biology and biomedicine, in particular to a large-area fluorescence imaging detection device.
Background
The technology of droplet digital polymerase chain reaction (ddPCR) is a method for absolutely quantifying nucleic acid molecules. Compared with quantitative PCR (qPCR), the method has the advantages of incomparable precision, accuracy and sensitivity; meanwhile, ddPCR eliminates the dependence on a quantitative standard curve, and improves the tolerance capability to an amplification inhibitor; therefore, ddPCR is called third generation PCR technology.
The working principle of ddPCR is to carry out microdroplet treatment on reaction liquid before PCR amplification, namely, the reaction liquid containing a nucleic acid template is dispersed into tens of thousands of nano-liter-level micro-droplets, each micro-droplet does not contain or contains one to a plurality of nucleic acid target molecules to be detected, and each micro-droplet is used as an independent PCR reaction unit. After PCR amplification, the micro-droplets containing the nucleic acid target molecules to be detected generate fluorescence signals, and the micro-droplets not containing the nucleic acid target molecules to be detected do not generate fluorescence signals. And finally, calculating the initial concentration or copy number of the nucleic acid target molecules to be detected according to the Poisson distribution principle and the proportion of the positive micro-droplets.
The steps of detecting target nucleic acid in a sample by utilizing the conventional commercial micro-droplet digital PCR device are as follows: 1) preparing ddPCR reaction liquid, and adding the ddPCR reaction liquid into the micro-droplet generation card; 2) preparing micro-droplets, wherein target nucleic acid is randomly distributed in each micro-droplet; 3) carrying out conventional PCR amplification after membrane sealing; 4) reading the fluorescence intensity of each micro-droplet one by utilizing a flow detection technology; 5) and (3) automatically distinguishing the negative/positive micro-droplets by using system software, counting the number of the negative/positive micro-droplets, and calculating the absolute copy number of the target nucleic acid according to the proportion of the positive micro-droplets and the Poisson distribution statistical principle. The existing micro-droplet fluorescence detection technology has the following problems: 1) the fluorescence intensity of each micro-droplet is read one by utilizing a flow detection technology, the speed is low, the time consumption is long, and the flux is low; 2) the optical path hardware of the flow detection technology is complex, and the multiple digital PCR detection (more than 4 times) is difficult to realize; 3) the flow detection technology detects the fluorescence intensity of the end point of the micro-droplet, and the qPCR detection of the micro-droplet cannot be realized; 4) the two transfer processes of the micro-droplets (the generated micro-droplets are transferred into a PCR instrument and the micro-droplets are transferred into a flow detector after PCR reaction) cause the loss of the micro-droplets; 5) the centrifuge tube containing the micro-droplets is opened for a plurality of times, which may cause cross contamination.
In order to solve the above problems, the skilled in the art has developed a digital PCR technique with single-layer tiled microdroplet array, for example, chinese patent CN104450891A discloses a method for generating single-layer tiled microdroplet array on the bottom of a flat-bottom container by using an interface vertical vibration method. The technical method has the following advantages: 1) the single-layer tiled micro-droplet array is suitable for large-area imaging fluorescence imaging detection by using an arrayed photoelectric sensor, and has high detection speed and detection flux; 2) the PCR amplification state of the micro-droplets can be monitored in real time by using continuous fluorescence imaging detection, so that the qPCR detection of the micro-droplets is realized, and the high sensitivity and specificity are realized; 3) by adding an excitation light source and an optical filter, multiple digital PCR (more than 4 times) can be realized; 4) the generation of micro-droplets, the PCR amplification and the fluorescence imaging detection of the micro-droplets are integrally completed, and the possibility of micro-droplet loss and cross contamination does not exist; 5) the micro-droplets spread in a single layer have larger temperature control area and smaller thermal inertia, and can realize rapid PCR temperature control circulation.
In order to detect target nucleic acid by using the single-layer tiled micro-droplet array, on one hand, temperature-controlled cyclic amplification needs to be performed on micro-droplets, and on the other hand, fluorescence excitation and fluorescence imaging detection needs to be performed on the single-layer tiled micro-droplet array. In addition, in order to improve the imaging detection efficiency, the single-layer tiled micro-droplet array or the fluorescence imaging detection device needs to perform one-dimensional or two-dimensional motion so as to acquire a large-area fluorescence image in a scanning mode. However, the existing fluorescence imaging detection devices, such as fluorescence microscopes, cannot meet the above detection requirements.
Disclosure of Invention
In view of the above, the present application discloses a large-area fluorescence imaging detection apparatus.
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection apparatus with a reflecting mirror, which reduces the size of the fluorescence detection apparatus, and shortens the distance between the optical imaging lens and the stage, thereby improving the fluorescence light collection efficiency.
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection apparatus with a light-limiting aperture plate, which limits the excitation light to only excite the fluorescence imaging region, and avoids the fluorescence quenching caused by long-time excitation light exposure in other regions.
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection apparatus with a temperature control loop stage, which performs real-time fluorescence imaging detection on a single-layer tiled micro droplet array while performing real-time temperature control loop operation.
It is a primary objective of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection apparatus with simple optical path hardware, which can integrate a multi-color composite light source or multiple monochromatic light sources, an excitation light filter assembly and a fluorescence filter assembly to realize multiple digital PCR detection (more than 4 times).
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art, and to provide a fluorescence imaging detection apparatus provided with a motion device, wherein the motion device can drive the objective table or the optical path system to move in at least one direction of freedom of motion, so as to realize single-layer tiled micro droplet array large-area fluorescence imaging detection.
In order to achieve the purpose, the following technical scheme is adopted in the application:
1. the large-area fluorescence imaging detection device is characterized by comprising a fluorescence excitation light mechanism, an excitation light reflecting mechanism and a fluorescence projecting mechanism,
the fluorescence excitation light mechanism comprises an excitation light source component, a collimating lens group, an excitation light filter component and a dichroic mirror which are sequentially arranged, and light emitted by the excitation light source component sequentially passes through the collimating lens group, the excitation light filter component and the dichroic mirror to form a fluorescence excitation light path;
the exciting light reflection mechanism comprises a light limiting hole plate and an object stage which are sequentially arranged, and light rays emitted by the dichroic mirror form an exciting light reflection light path through the light limiting hole plate and the object stage;
the fluorescence projection mechanism comprises a fluorescence optical filter component, an optical imaging lens and an image sensor which are arranged in sequence; the light reflected by the objective table passes through the light limiting hole plate, the dichroic mirror, the fluorescent filter assembly, the optical imaging lens and the image sensor to form a fluorescent projection light path;
the light rays entering the dichroic mirror form an angle of 45 degrees +/-10 degrees with the mirror surface of the dichroic mirror, and the light rays reflected by the dichroic mirror form an angle of 0 degrees +/-10 degrees with the normal line of the objective table;
the exciting light reflection light path is coaxial with the fluorescence projection light path.
2. The large-area fluorescence imaging detection device according to item 1, further comprising a mirror located between the excitation light filter assembly and the dichroic mirror; the light emitted by the exciting light filter component is emitted to the dichroic mirror through the reflecting mirror.
3. The large-area fluorescence imaging detection device according to item 2, wherein the light emitted by the excitation light source assembly forms an angle of 45 ° ± 10 ° with the mirror surface of the reflector;
the reflecting mirror is arranged in parallel with the dichroic mirror;
the reflector is a plane reflector.
4. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the excitation light filter assembly comprises a first motor, a first turntable and an excitation light filter, the first motor is connected with the first turntable, and the excitation light filter is disposed on the first turntable.
5. The large-area fluorescence imaging detection device according to item 4, wherein one or more first mounting holes are uniformly formed in the first turntable with the center of the first turntable as a symmetry center, and the excitation light filter is disposed in the first mounting holes;
the out-of-band inhibition OD of the exciting light filter is more than or equal to 5.
6. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the excitation light source assembly comprises a second motor, a second turntable and an excitation light source, the second motor is connected with the second turntable, and the excitation light filter is arranged on the second turntable.
7. The large-area fluorescence imaging detection device according to claim 6, wherein the center of the second turntable is taken as a symmetry center, the second turntable is uniformly provided with more than two second mounting holes, and the excitation light source is arranged in the second mounting holes;
the excitation light source is one or more than two of a monochromatic light source, a wide-spectrum light source or a multicolor composite light source.
8. The large-area fluorescence imaging detection apparatus according to claim 1 or 2, further comprising a dichroic mirror assembly, wherein the dichroic mirror assembly includes a third motor, a third turntable, and the dichroic mirror, the third motor is connected to the third turntable, and the dichroic mirror is disposed on the third turntable.
9. The large-area fluorescence imaging detection device according to claim 8, wherein two or more third mounting holes are uniformly formed in the third turntable with the center of the third turntable as a symmetry center, and the dichroic mirror is disposed in the third mounting holes;
the dichroic mirror is a single-pass single-reflection type dichroic mirror or a multi-pass multi-reflection type dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror is more than 0.9, and the OD of the out-of-channel inhibition is more than or equal to 3.
10. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the fluorescence filter assembly comprises a fourth motor, a fourth turntable and the fluorescence filter, the fourth motor is connected with the fourth turntable, and the fluorescence filter is arranged on the fourth turntable.
11. The large-area fluorescence imaging detection apparatus according to item 10, wherein two or more fourth mounting holes are uniformly provided in the fourth turntable with the center of the fourth turntable as a symmetry center, and the fluorescence filter is provided in the fourth mounting hole;
the transmittance or reflectivity in the channel of the fluorescent filter is more than 0.9, and the OD of the out-of-channel inhibition is more than or equal to 5.
12. The large-area fluorescence imaging detection device according to claim 1 or 2, further comprising a motion mechanism, wherein the motion mechanism comprises a first motion mechanism and a second motion mechanism, the first motion mechanism is connected with the image sensor, and the first motion mechanism is used for controlling the image sensor to move relative to the optical imaging lens; the second motion mechanism is connected with the object stage, so that the second motion mechanism is used for controlling the object stage to move relative to the light limiting hole plate.
13. The large-area fluorescence imaging detection device according to item 1, wherein the image sensor is one of a complementary metal oxide semiconductor, a charge coupled device or an array photosensor;
the image sensor pixel size is less than 12.4 microns, and the dynamic range is larger than 4700 dB.
14. The large area fluorescence imaging detection device of claim 1 or 2, wherein the collimating lens group comprises a spherical lens and/or an aspherical lens.
15. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the light limiting hole plate is provided with a plurality of light limiting holes, the shape of each light limiting hole is one of a circle, a rectangle, a triangle, a rhombus or other polygons, and the area of each light limiting hole is not less than 16mm2。
16. The large-area fluorescence imaging detection device according to item 1 or 2, wherein a temperature control mechanism is provided on the stage, and the temperature control mechanism is used for setting the reaction precision temperature of the sample to be detected and controlling the cyclic temperature control reaction.
17. The large-area fluorescence imaging detection device according to item 1 or 2, wherein the optical imaging lens is automatically or manually continuously adjusted steplessly within a magnification of 0.1 to 5.
Compared with the prior art, the large-area fluorescence imaging detection device disclosed by the application has the beneficial effects that: by arranging the reflector, the size of the fluorescence detection device is reduced, the distance between the optical imaging lens and the objective table is shortened, and the fluorescence light-collecting efficiency is improved; by arranging the light limiting hole plate, the exciting light is limited to only excite the fluorescence imaging area, so that fluorescence quenching caused by long-time exciting light exposure of other areas is avoided; the objective table with the temperature control circulation is used for realizing real-time temperature control circulation operation of the single-layer tiled micro-droplet array and simultaneously carrying out real-time fluorescence detection on the single-layer tiled micro-droplet array; the simple optical path hardware system can integrate a single multicolor composite light source or a plurality of monochromatic light sources, an excitation light filter component and a fluorescence filter component to realize multiple digital PCR detection (more than 4 times); the moving device can drive the objective table or the light path system to move in at least one movement freedom degree direction, so that the single-layer tiled micro-droplet array large-area fluorescence imaging detection is realized.
Drawings
The drawings are included to provide a further understanding of the application and are not to be construed as limiting the application. Wherein:
fig. 1 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
Fig. 2 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
Fig. 3 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
FIG. 4 is a schematic structural diagram of a collimating lens assembly.
Fig. 5 is a schematic structural diagram of an excitation light filter assembly, which includes a turntable and a plurality of excitation light filters, wherein mounting holes are distributed along the circumferential direction.
Fig. 6 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
Fig. 7 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
Fig. 8 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
FIG. 9 is a schematic diagram of a large-area fluorescence imaging detection device provided with a motion device connected to an optical path system.
Fig. 10 is a schematic structural diagram of a large-area fluorescence imaging detection device in the present application.
Fig. 11 is a graph of experimental results for a single-layer tiled array of microdroplets in example 1.
Fig. 12 is a graph of experimental results for a single-layer tiled array of microdroplets in example 2.
Fig. 13 is a graph of the results of a single-layer tiled microdroplet array in example 3.
Wherein, the reference numbers:
1-excitation light source component, 2-collimating lens group, 3-excitation light filter component, 4-reflector, 5-dichroic mirror, 6-light limiting orifice plate, 7-objective table, 8-fluorescence filter, 9-optical imaging lens, 10-image sensor, 11-fluorescence filter component, 12-first motor, 13-first turntable, 14-excitation light filter, 15-excitation light source, 16-spherical lens, 17-aspherical lens, 18-second motor, 19-second turntable, 20-third motor, 21-third turntable, 22-first movement mechanism, 23-fifth transmission mechanism, 24-fourth motor, 25-fourth turntable, 26-second movement mechanism and 27-sixth transmission mechanism, 28-first mounting hole.
Detailed Description
The following description of the exemplary embodiments of the present application, taken in conjunction with the accompanying drawings, includes various details of the embodiments of the application for the understanding of the same, which are to be considered exemplary only. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application. Also, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.
In the present application, as shown in fig. 1, a large-area fluorescence imaging detection apparatus disclosed in the present application includes a fluorescence excitation light mechanism, an excitation light reflection mechanism, and a fluorescence projection mechanism, where the fluorescence excitation light mechanism includes an excitation light source 15, a collimating lens group 2, an excitation light filter 14, a reflecting mirror 4, and a dichroic mirror 5, which are sequentially arranged; the light emitted by the excitation light source 15 sequentially passes through the collimating lens group 2, the excitation light filter 14, the reflecting mirror 4 and the dichroic mirror 5 to form a fluorescence excitation light path;
the exciting light reflection mechanism comprises a light limiting hole plate 6 and an object stage 7 which are sequentially arranged, and light rays emitted by the dichroic mirror 5 form an exciting light reflection light path through the light limiting hole plate 6 and the object stage 7;
the fluorescence projection mechanism comprises a fluorescence filter 8, an optical imaging lens 9 and an image sensor 10 which are arranged in sequence; the light reflected by the objective table 7 passes through the light limiting hole plate 6, the dichroic mirror 5, the fluorescent filter 8, the optical imaging lens 9 and the image sensor 10 to form a fluorescent projection light path; the light rays entering the dichroic mirror 5 form an angle of 45 degrees +/-10 degrees with the mirror surface of the dichroic mirror 5, and the light rays reflected by the dichroic mirror 5 form an angle of 0 degrees +/-10 degrees with the normal line of the objective table 7; the exciting light reflection light path is coaxial with the fluorescence projection light path.
The collimating lens group 2 is arranged right below the excitation light source 15, the excitation light filter 14 is fixedly arranged right below the collimating lens group 2, the reflecting mirror 4 is fixedly arranged right below the excitation light filter 14, the reflecting mirror 4 is parallel to the dichroic mirror 5, and the reflecting mirror 4 and the dichroic mirror 5 are positioned at the same horizontal height. And a limiting hole is fixedly arranged under the dichroic mirror 5, and an objective table 7 is arranged under the limiting hole. A fluorescent filter 8 is arranged right above the dichroic mirror 5, an optical imaging lens 9 is arranged right above the fluorescent filter 8, and an image sensor 10 is arranged right above the optical imaging lens 9.
The collimating lens group 2 is disposed between the excitation light source 15 and the excitation light filter 14, and is configured to collimate excitation light emitted by the excitation light source 15 to form a parallel light beam. As shown in fig. 4, the collimating lens group 2 includes a spherical lens 16 and an aspherical lens 17, and preferably, the collimating lens group 2 has a working distance of not more than 5cm to increase the amount of light entering the fluorescence excitation light path.
The excitation light source 15 may be one of a monochromatic light source, a broad spectrum light source, or a polychromatic composite light source.
The reflecting mirror 4 is a plane reflecting mirror 4, is fixedly disposed between the excitation light filter 14 and the dichroic mirror 5, and is disposed at an angle of 45 ± 10 degrees with respect to the light emitted by the excitation light source 15, and is configured to reflect the light emitted by the excitation light source 15 to the dichroic mirror 5. By arranging the reflector, the size of the fluorescence detection device is reduced, the distance between the optical imaging lens 9 and the objective table 7 is shortened, and the fluorescence light collection efficiency is improved.
The OD of the out-of-band inhibition of the exciting light filter 14 is more than or equal to 5.
The dichroic mirror 5 is a single-pass single-reflection type dichroic mirror or a multi-pass multi-reflection type dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror 5 is more than 0.9, and the OD of the out-of-channel inhibition is more than or equal to 3.
The transmittance or reflectivity in the channel of the fluorescent filter 8 is more than 0.9, and the OD of the out-of-channel inhibition is more than or equal to 5.
The image sensor 10 has a pixel size of less than 12.4 microns and a dynamic range of greater than 4700 dB. Dynamic range, i.e., the maximum output signal level that a single pixel of the image sensor can produce (full well capacity) divided by the ratio of the signal level that would be produced even if the pixel had no incident light (noise floor).
The light limiting hole plate 6 is provided with a plurality of light limiting holes, the shape of each light limiting hole is one of circular, rectangular, triangular, rhombic or other polygons, and the area of each light limiting hole is not less than 16mm2. The shape of the light limiting hole can be determined according to practical application and requirements, and the area of the light limiting hole is not less than 16mm2. The light limiting hole plate 6 is used for limiting exciting light to only irradiate a fluorescence imaging area, and fluorescence quenching caused by long-time exciting light exposure of other areas is avoided.
In the present application, as shown in fig. 10, an excitation light source 15, a collimating lens group 2, an excitation light filter 14, and a dichroic mirror 5 are located at the same horizontal height, and light emitted from the excitation light source 15 passes through the collimating lens group 2 and the excitation light filter 14 and is incident on the dichroic mirror, and a mirror surface of the dichroic mirror is 45 ° ± 10 ° with respect to light emitted from the excitation light filter 14.
In the present application, as shown in fig. 2 and fig. 5, the large-area fluorescence imaging detection apparatus disclosed in the present application, the excitation light filter assembly 3 includes a first motor 12, a first rotating disk 13 and an excitation light filter 14, the first motor 12 is connected to the first rotating disk 13, and the excitation light filter 14 is disposed on the first rotating disk 13. One or more first mounting holes 28 are uniformly formed in the first rotating disk 13 by taking the center of the first rotating disk 13 as a symmetrical center, and the excitation light filter 14 is arranged in each first mounting hole 28; the OD of the out-of-band inhibition of the exciting light filter 14 is more than or equal to 5.
The number of the first mounting holes 28 on the first rotary disk 13 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and size of the first mounting holes 28 may be determined according to actual needs. The excitation light filters 14 on the plurality of first mounting holes 28 may be the excitation light filters 14 having the same out-of-band rejection, and may be the excitation light filters 14 having different out-of-band rejections. The relative installation positions and the number of the excitation light filters 14 of different types can be determined according to actual needs.
The plane of the excitation light filter 14 is perpendicular to the light emitted from the excitation light source 15.
The first motor 12 may be one of a stepping servo motor, a dc servo motor, or an ac servo motor. The first motor 12 drives the first rotating disc 13 to rotate through the first transmission mechanism, so that the required excitation light filter 14 rotates to a specified position, the excitation light filters 14 are respectively configured in the fluorescence excitation light path in a switchable manner, the out-of-band inhibition OD of the excitation light filter 14 is larger than or equal to 5, and monochromatic light output of the excitation light source assembly 1 with different wavelengths is realized.
In this application, as shown in fig. 3, the large-area fluorescence imaging detection apparatus disclosed in this application, the excitation light source assembly 1 includes a second motor 18, a second turntable 19 and an excitation light source 15, the second motor 18 is connected with the second turntable 19, and the excitation light filter is disposed on the second turntable 19. The center of the second turntable 19 is taken as a symmetry center, more than two second mounting holes are uniformly formed in the second turntable 19, and the excitation light sources 15 are arranged in the second mounting holes;
the excitation light source 15 is one or more of a monochromatic light source, a wide-spectrum light source or a multicolor composite light source.
The number of the second mounting holes on the second rotary table 19 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and size of the second mounting holes may be determined according to actual needs. The excitation light sources 15 on the second mounting holes may all be a monochromatic light source, a wide-spectrum light source or a multicolor composite light source, or may be a combination of a monochromatic light source, a wide-spectrum light source and a multicolor composite light source. When the excitation light sources 15 are a monochromatic light source, a wide-spectrum light source, or a multicolor composite light source, the relative installation positions and the number of the excitation light sources 15 can be determined according to actual needs.
The second motor 18 drives the second turntable 19 to rotate through a second transmission mechanism, so as to rotate the required excitation light source 15 to a specified position, and the excitation light source 15 is respectively configured in the fluorescence excitation light path in a switchable manner, thereby realizing output of monochromatic excitation light of different wavelengths of a multi-color and multi-type composite light source.
The second motor 18 may be one of a stepping servo motor, a dc servo motor, or an ac servo motor. When the excitation light source 15 adopts a multicolor composite light source, the multicolor composite light source and the excitation light filter component 3 generate monochromatic excitation light, so that the monochromatic excitation light with multiple wavelengths can be provided at a lower cost, and the function of multiplex digital PCR is realized.
When the excitation light source 15 is a plurality of monochromatic light sources, the implementation of exciting the single-layer tiled microdroplet array by the excitation light with different wavelengths is as follows: the second motor 18 drives the second turntable 19 to make the monochromatic light sources thereon be respectively configured into the fluorescence excitation light path in a switchable manner, and emit monochromatic excitation light with different wavelengths; the collimating lens group 2 collimates monochromatic light emitted by the monochromatic light source to form parallel light beams, the excitation light filter assembly 3 includes a first motor 12, a first rotating disc 13 with a plurality of first mounting holes 28 distributed along the circumferential direction, and a plurality of excitation light filters 14, the first motor 12 drives the first rotating disc 13 to make the excitation light filters 14 thereon respectively arranged in the fluorescence excitation light path in a switchable manner, so as to filter out the wavelength of the unwanted excitation light.
In the present application, as shown in fig. 6, for the large-area fluorescence imaging detection apparatus disclosed in the present application, the apparatus further includes a dichroic mirror 5 assembly, the dichroic mirror 5 assembly includes a third motor 20, a third turntable 21 and the dichroic mirror 5, the third motor 20 is connected to the third turntable 21, and the dichroic mirror 5 is disposed on the third turntable 21. With the center of the third turntable 21 as a symmetry center, two or more third mounting holes are uniformly formed in the third turntable 21, and the dichroic mirror 5 is arranged in each third mounting hole;
the dichroic mirror 5 is a single-pass single-reflection type dichroic mirror or a multi-pass multi-reflection type dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror 5 is more than 0.9, and the OD of the out-of-channel inhibition is more than or equal to 3. Out-of-band rejection refers to the ability of a module to reject optical signals outside the range of the effective occupied band.
The number of the third mounting holes on the third rotary table 21 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and size of the third mounting holes may be determined according to actual needs. The dichroic mirrors 5 on the third mounting holes may be single-pass single-reflection dichroic mirrors or multi-pass multi-reflection dichroic mirrors, or may be a combination of single-pass single-reflection dichroic mirrors or multi-pass multi-reflection dichroic mirrors. When the dichroic mirror 5 is a single-pass single-reflection dichroic mirror or a multi-pass multi-reflection dichroic mirror, the relative installation positions and the number of the two dichroic lenses can be determined according to actual needs.
The third motor 20 drives the third turntable 21 to rotate through a third transmission mechanism, so as to rotate the required dichroic lens to a specified position, and the dichroic lenses are respectively configured in the fluorescence excitation light path in a switchable manner. When the single-pass single-reflection dichroic mirror is disposed at an angle of 45 ° ± 10 ° with respect to the fluorescence excitation light path and the fluorescence transmission light path, the single-pass single-reflection dichroic mirror reflects the excitation light of short wavelength, enters the stage 7 at a vertical angle, and simultaneously, transmits the fluorescence of long wavelength to enter the image sensor 10.
When the dichroic mirror 5 is a multi-pass multi-reflection dichroic mirror, it is fixedly disposed in the fluorescence excitation light path. When the multi-pass multi-reflection dichroic mirror is arranged at an angle of 45 ° ± 10 ° with respect to the fluorescence excitation light path and the fluorescence transmission light path, the multi-pass multi-reflection dichroic mirror reflects excitation light of a plurality of wavelength bands, enters the stage 7 at a vertical angle, and simultaneously enters the image sensor 10 through fluorescence of a plurality of wavelength bands.
The third motor 20 may be one of a stepping servo motor, a dc servo motor, or an ac servo motor.
In the present application, as shown in fig. 7, for the large-area fluorescence imaging detection apparatus disclosed in the present application, the fluorescence filter assembly 11 includes a fourth motor 24, a fourth turntable 25 and the fluorescence filter 8, the fourth motor 24 is connected to the fourth turntable 25, and the fluorescence filter 8 is disposed on the fourth turntable 25. With the center of the fourth turntable 25 as a symmetry center, more than two fourth mounting holes are uniformly formed in the fourth turntable 25, and the fluorescent filters 8 are arranged in the fourth mounting holes;
the number of the fourth mounting holes on the fourth rotating disk 25 may be 1, 2, 3, 4, 5, 6, 7, etc., and the number and size of the fourth mounting holes may be determined according to actual needs.
The fourth motor 24 drives the fourth turntable 25 to rotate through a fourth transmission mechanism, so that the required fluorescent filter 8 is rotated to a specified position, the fluorescent filter 8 is configured in the fluorescent transmission light path in a switchable manner, the transmittance or reflectivity in the channel of the fluorescent filter 8 is greater than 0.9, and the out-of-channel inhibition OD is greater than or equal to 5, so as to filter out the unnecessary fluorescent wavelength.
The optical imaging lens 9 is arranged between the fluorescence filter assembly 11 and the image sensor 10, the magnification of the optical imaging lens can be continuously adjusted in a stepless manner within 0.1-5 x, and the optical imaging lens is used for collecting fluorescence emitted by the excited single-layer tiled micro-droplet array.
In the present application, as shown in fig. 8 and fig. 9, the large-area fluorescence imaging detection apparatus disclosed in the present application further includes a motion mechanism, the motion mechanism includes a first motion mechanism 22 and a second motion mechanism 26, the first motion mechanism 22 is connected to the image sensor 10, and the first motion mechanism 22 is configured to control the image sensor 10 to move relative to the optical imaging lens 9; the second movement mechanism 22 is connected to the object stage 7, so that the second movement mechanism 22 is used to control the object stage 7 to move relative to the light-limiting aperture plate 6.
The first movement mechanism 22 includes a fifth motor and a fifth transmission mechanism 23, the fifth transmission mechanism 23 is connected to the image sensor 10, and the fifth motor can drive the image sensor 10 to move in one-dimensional or two-dimensional direction through the fifth transmission mechanism 23, so as to realize the movement of the image sensor 10 in one or two movement freedom directions. Therefore, the one-dimensional or two-dimensional motion of the image sensor 10 in the horizontal X direction or the Y direction can be precisely controlled, and the one-dimensional or two-dimensional scanning detection of the micro-droplet arrays tiled at different hole bottoms of the perforated container by the fluorescence imaging detection device can be realized, so that the detection range is improved, and the large-area high-flux fluorescence imaging detection is realized.
The second movement mechanism 26 includes a sixth motor and a sixth transmission mechanism 27, the sixth transmission mechanism 27 is connected to the object stage 7, and the sixth motor can drive the object stage 7 to move in one-dimensional or two-dimensional direction through the sixth transmission mechanism 27, so as to realize the movement of the object stage 7 in one or two movement freedom directions. Therefore, the one-dimensional or two-dimensional motion of the object stage 7 in the horizontal X direction or the Y direction can be precisely controlled, and the one-dimensional or two-dimensional scanning detection of the micro-droplet arrays paved at different hole bottoms of the perforated container by the fluorescence imaging detection device can be realized, so that the detection range is improved, and the large-area high-flux fluorescence imaging detection is realized.
The fifth motor and the sixth motor can be one of a stepping servo motor, a direct current servo motor or an alternating current servo motor.
In the present application, the stage 7 is provided with a temperature control mechanism for reaction accurate temperature setting and cyclic temperature control reaction control of the sample to be measured. Multiple digital PCR can be realized by adding multiple fluorescent dyes or probes to a PCR reaction system and matching with different PCR reaction premixed solution and primers. Wherein the PCR multiplicity is the same as the type of fluorochrome or probe added, e.g., quadruple digital PCR requires four fluorochromes or probes.
In the present application, the image sensor 10 is one of a Complementary Metal Oxide Semiconductor (CMOS), a Charge Coupled Device (CCD), or an array photosensor, and the image sensor 10 has a pixel size of less than 12.4 microns and a dynamic range of greater than 4700dB for converting optical signals to image signals for subsequent analysis.
Examples
The experimental methods used in the following examples are all conventional methods, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Adopting the large-area fluorescence imaging detection device shown in fig. 8, starting an excitation light source (the excitation light source is a self-developed high-power multicolor composite LED light source), placing an open container provided with a single-layer tiled micro-droplet (the micro-droplet is an aqueous solution containing 0.4mg/mL rhodamine B) array on an object stage (zhulihan light), and adjusting the movement mechanism to enable a sample cell of the open container to be positioned in the center of a visual field; the excitation light source emits light, which is collimated by a collimating lens set (diameter 25mm, focal length 50mm) and enters an excitation light filter (the excitation light filter is a filter of 520nm-540 nm), and the excitation light passes through the excitation light filter set to filter out unnecessary wave bands and then is reflected by a reflector to enter a single-pass single-reflection dichroic mirror (total reflection at wavelength below 550 nm)Emitting light with a wavelength of 550nm or more), reflecting short-wavelength excitation light by a single-pass single-reflection type dichroic mirror, limiting the size and shape of a light spot by a light limiting hole plate (a circular hole with an aperture of 15mm), then irradiating the light spot to excite a single-layer tiled micro-droplet array in an open container, filtering unnecessary wave bands by a fluorescence filter (the fluorescence filter is a 554nm-576nm filter) after the micro-droplets pass through the light limiting hole plate and the single-pass single-reflection type dichroic mirror, collecting the light bands by an optical imaging lens, reaching an image sensor, converting the light bands into image signals, and detecting the fluorescence imaging of the micro-droplet array as shown in figure 11, wherein the imaging area of the micro-droplet array is 64mm2。
Example 2
The steps in example 1 were used to perform fluorescence imaging detection on the single-layer tiled microdroplet array, where the microdroplet was an aqueous solution containing 200mM green fluorescein, the excitation light was a multicolor composite light source, the excitation light filter was a 440nm-460nm filter, and the fluorescence filter was a 500nm-550nm filter. The experimental result of fluorescence imaging detection of the micro-droplet array is shown in FIG. 12, in which the imaging area of the micro-droplet array is 64mm2。
Example 3
Fluorescence imaging detection of a single-layer tiled array of microdroplets, comprising: 2. mu.L of 10 × reaction buffer, 1.5. mu.L of 10mM forward primer (5'-GGACTC TGGACA TGCAAGAT-3'), 1.5. mu.L of 10mM reverse primer (5'-ATACCC TTC TTAACACCT GG-3'), 0.5. mu.L of 10mM Taqman probe (5 '-FAM-GTATCAATTTAGAGAAAA CGC TCT GAA GGG-BHQ 1-3'), 1. mu.L of 4mM dNTPmix, 20.4. mu.L of 100mM MgCl20, 0.4. mu.L of 2U/. mu.L TransScript DNA polymerase, 1. mu.L of Saccharomyces cerevisiae HansenATCC 204508/S288C genomic DNA (10-fold dilution), and 11.7. mu.L of deionized water. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s and annealing extension at 55 ℃ for 40s for 40 cycles. The exciting light is a multicolor composite light source, the exciting light filter is a 440nm-460nm filter, and the fluorescent filter is a 500nm-550nm filter. The experimental result of fluorescence imaging detection of the micro-droplet array is shown in FIG. 13, in which the imaging area of the micro-droplet array is 64mm2。
Although the embodiments of the present application have been described above with reference to the accompanying drawings, the present application is not limited to the above-described embodiments and application fields, and the above-described embodiments are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto and changes may be made without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. The large-area fluorescence imaging detection device is characterized by comprising a fluorescence excitation light mechanism, an excitation light reflecting mechanism and a fluorescence projecting mechanism,
the fluorescence excitation light mechanism comprises an excitation light source component, a collimating lens group, an excitation light filter component and a dichroic mirror which are sequentially arranged, and light emitted by the excitation light source component sequentially passes through the collimating lens group, the excitation light filter component and the dichroic mirror to form a fluorescence excitation light path;
the exciting light reflection mechanism comprises a light limiting hole plate and an object stage which are sequentially arranged, and light rays emitted by the dichroic mirror form an exciting light reflection light path through the light limiting hole plate and the object stage;
the fluorescence projection mechanism comprises a fluorescence optical filter component, an optical imaging lens and an image sensor which are arranged in sequence; the light reflected by the objective table passes through the light limiting hole plate, the dichroic mirror, the fluorescent filter assembly, the optical imaging lens and the image sensor to form a fluorescent projection light path;
the light rays entering the dichroic mirror form an angle of 45 degrees +/-10 degrees with the mirror surface of the dichroic mirror, and the light rays reflected by the dichroic mirror form an angle of 0 degrees +/-10 degrees with the normal line of the objective table;
the exciting light reflection light path is coaxial with the fluorescence projection light path.
2. The large area fluorescence imaging detection device of claim 1, further comprising a mirror between the excitation light filter assembly and the dichroic mirror; the light emitted by the exciting light filter component is emitted to the dichroic mirror through the reflecting mirror.
3. The large-area fluorescence imaging detection device according to claim 2, wherein the light emitted from the excitation light source assembly forms an angle of 45 ° ± 10 ° with the mirror surface of the reflector;
the reflecting mirror is arranged in parallel with the dichroic mirror;
the reflector is a plane reflector.
4. The large-area fluorescence imaging detection device according to claim 1 or 2, wherein the excitation light filter assembly comprises a first motor, a first turntable, and an excitation light filter, the first motor is connected to the first turntable, and the excitation light filter is disposed on the first turntable.
5. The large-area fluorescence imaging detection device according to claim 4, wherein one or more first mounting holes are uniformly formed in the first turntable with the center of the first turntable as a symmetry center, and the excitation light filter is disposed in the first mounting holes;
the out-of-band inhibition OD of the exciting light filter is more than or equal to 5.
6. The large-area fluorescence imaging detection device according to claim 1 or 2, wherein the excitation light source assembly comprises a second motor, a second turntable, and an excitation light source, the second motor is connected to the second turntable, and the excitation light filter is disposed on the second turntable.
7. The large-area fluorescence imaging detection device according to claim 6, wherein two or more second mounting holes are uniformly formed in the second turntable with the center of the second turntable as a symmetry center, and the excitation light source is disposed in the second mounting holes;
the excitation light source is one or more than two of a monochromatic light source, a wide-spectrum light source or a multicolor composite light source.
8. The large area fluorescence imaging detection apparatus of claim 1 or 2, further comprising a dichroic mirror assembly, the dichroic mirror assembly comprising a third motor, a third turntable, and the dichroic mirror, the third motor being connected to the third turntable, the dichroic mirror being disposed on the third turntable.
9. The large-area fluorescence imaging detection device according to claim 8, wherein two or more third mounting holes are uniformly formed in the third turntable with the center of the third turntable as a symmetry center, and the dichroic mirror is disposed in the third mounting holes;
the dichroic mirror is a single-pass single-reflection type dichroic mirror or a multi-pass multi-reflection type dichroic mirror; the transmittance or reflectivity in the channel of the dichroic mirror is more than 0.9, and the OD of the out-of-channel inhibition is more than or equal to 3.
10. The large-area fluorescence imaging detection device according to claim 1 or 2, wherein the fluorescence filter assembly comprises a fourth motor, a fourth turntable, and the fluorescence filter, the fourth motor is connected to the fourth turntable, and the fluorescence filter is disposed on the fourth turntable.
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